Histological evaluation of the distribution of systemic AA-amyloidosis in nine domestic shorthair cats

Amyloidosis is a group of protein-misfolding disorders characterized by the accumulation of amyloid in organs, both in humans and animals. AA-amyloidosis is considered a reactive type of amyloidosis and in humans is characterized by the deposition of AA-amyloid fibrils in one or more organs. In domestic shorthair cats, AA-amyloidosis was recently reported to be frequent in shelters. To better characterize this pathology, we report the distribution of amyloid deposits and associated histological lesions in the organs of shelter cats with systemic AA-amyloidosis. AA-amyloid deposits were identified with Congo Red staining and immunofluorescence. AA-amyloid deposits were then described and scored, and associated histological lesions were reported. Based on Congo Red staining and immunofluorescence nine shelter cats presented systemic AA-amyloidosis. The kidney (9/9), the spleen (8/8), the adrenal glands (8/8), the small intestine (7/7) and the liver (8/9) were the organs most involved by amyloid deposits, with multifocal to diffuse and from moderate to severe deposits, both in the organ parenchyma and/or in the vascular compartment. The lung (2/9) and the skin (1/8) were the least frequently involved organs and deposits were mainly focal to multifocal, mild, vascular and perivascular. Interestingly, among the organs with fibril deposition, the stomach (7/9), the gallbladder (6/6), the urinary bladder (3/9), and the heart (6/7) were reported for the first time in cats. All eye, brain and skeletal muscle samples had no amyloid deposits. An inflammatory condition was identified in 8/9 cats, with chronic enteritis and chronic nephritis being the most common. Except for secondary cell compression, other lesions were not associated to amyloid deposits. To conclude, this study gives new insights into the distribution of AA-amyloid deposits in cats. A concurrent chronic inflammation was present in almost all cases, possibly suggesting a relationship with AA-amyloidosis.


Introduction:
The objective of this study can be written in a better way and more interesting.
-Authors response: thank you for your comment.We changed the final part of the introduction according to your comment.
The Authers not highlighting the lack of information about the histopathological findings that could be the objective of this study.
-Authors response: we also added this missing information in the introduction.
In general, the introduction has sufficient information about the nature of amyloidosis and its prevalence.

Material and Methods:
Materials and methods were written subtitled in a good way but why not talk about precautions taken during collecting testes samples?
-Authors response: thank you for this comment, we added more details on how sample collection was performed.
Consider providing more details on specific staining techniques, reagents and equipment used.
-Authors response: we added information on Congo Red staining reagent.In our opinion, the rest of the materials and methods description is detailed.

Results:
The results are presented clearly and concisely, with appropriate tables and figures and statistical analysis.
It will be more beneficial to include additional representative images of histopathological findings to enhance the visual understanding of the results.
-Authors response: thank you for your comments.We added one image of a tongue to better show the architecture of the tissue and the localization of the amyloid.In our opinion, the included pictures are already numerous and the most representative for amyloid distribution.Although, we added symbols and labels to better identify the structures and we also improved the legends with more detailed descriptions to guide the reader.

Discussions:
The discussion provides a comprehensive interpretation of the result in the context of existing knowledge.
-Authors response: thank you for your comment.

Conclusion:
The conclusion succinctly summarizes the main findings and their implications.
It would be helpful to include suggestions for future research directions.
-Authors response: thank you for these comments.We added one sentence to suggest possible further investigations.

References:
The references are recent and up to date, adding to the currency of the paper.
The references are highly relevant to the topic matter being discussed.
The author appropriately draws connections between the references and their research.
-Authors response: thank you for your comment.

Writing style:
The writing style is clear and concise, making it easy to follow the author's idea.
Some parts could benefit from additional organization for improved readability.
-Authors response: thank you for your comment.We rephrased some sentences trying to improve the readability.

Additional Comments:
The study represents a valuable contribution to the field of hitopathology and provides novel insights into amyloidosis in cats.

Reviewer #2:
Dear Sir, Regarding your manuscript entitled "Histological evaluation of the distribution of systemic AAamyloidosis in nine domestic shorthair cats," I would like to express my opinion.In my personal view, the work is an excellent piece of research that sheds descriptive light on AA-amyloidosis in domestic shorthair cats.I have a few comments on the manuscript, mostly regarding the quality of its figures.I kindly request your patience as I provide my feedback: -For all figures, it would be beneficial to include micrometer measurements to assist in size estimation.Additionally, enhancing the photographs would help identify the described colorations (e.g., the blue DAPI coloration is not apparent in Fig. 1).In my humble opinion, including additional H&E staining pictures would greatly contribute to proper comparison and description of the individual tissue structures.
-Authors response: thank you for your comment.We added a scale bar in the pictures, included a H&E picture of the tongue, adjusted the contrast of some images and changed the plate for spleen pictures, to better represent amyloid distribution.In our opinion, new H&E images give no additional details because they are not representative of amyloid presence and distribution.Since the images are already numerous, we preferred instead to better explain the Congo Red pictures improving the legends and adding labels to help the reader.We hope you can find it useful.
-In Fig. 1b, the inset is not distinguishable from the original picture.
-Authors response: thank you for your comment.We corrected Fig. 1 b to highlight the inset.
Finally, I believe the work is worthy of publication once these minor corrections have been taken into consideration.Thank you very much.Best regards,